Method for inducing angiogenesis by electrical stimulation of muscles

ABSTRACT

The present invention discloses a method for angiogenesis. The method comprises the steps of electrically stimulating a muscle below the threshold for muscle contraction.

This application is a continuation of U.S. patent application Ser. No.09/858,036, filed May 15, 2001, which claims priority to U.S.provisional application Ser. No. 60/204,376, filed on May 16, 2000, thedisclosures of which are incorporated herein by reference.

FIELD OF INVENTION

The present invention relates generally to the field of angiogenesis.More particularly, the present invention provides a method for inducingangiogenesis by electrical stimulation.

DISCUSSION OF RELATED ART

Arterial occlusive diseases cause serious ischemia in various organs,such as the heart, brain, and leg. Therapeutic angiogenesis is thoughtto be beneficial for such conditions (Pepper, 1997, Arterioscler ThrombVasc Biol., 17:605-619). Local administration of recombinant angiogenicgrowth factors, such as basic fibroblast growth factor (bFGF) andvascular endothelial growth factor (VEGF), salvaged ischemic areas ofmyocardium and hindlimb in animal models (Baffour et al., 1992, J VascSurg., 16:181-191; Takeshita et al., 1994, J Clin Invest., 93:662-670;Hariwala et al., 1996, J Surg Res., 63:77-82; Banai et al., 1994,Circulation. 89:2183-2189; Yanagisawa-Miwa, 1992, Science,257:1401-1403). However, the clinical application requires large amountsof these recombinant proteins and is not feasible at this time. Insteadof recombinant proteins, use of gene therapy, i.e., in vivo transfectionof angiogenic growth factor genes, has been attempted to treat thesediseases. In particular, the clinical trial of intramuscular genetransfer of naked plasmid DNA encoding human VEGF₁₆₅ is progressing inthe United States for the treatment of ischemic limbs (Baumgartner etal. Circulation, 1998, 97:1114-1123).

VEGF, a dimeric endothelial cell (EC)-specific growth factor, is thoughtto be a principal angiogenic factor that stimulates migration,proliferation, and expression of various genes in endothelial cells(ECs; Leung et al., 1989, Science, 246:1306-1309; Plate et al., 1992,Nature, 359:845-848; Ferrara et al., 1992, Endocrinol Rev., 13:18-32).VEGF is synthesized by cells around vasculature and affects ECs as aparacrine factor. The expression of VEGF is upregulated by hypoxia andvarious cytokines.

Although some of the factors involved in angiogenesis have beenidentified, no simple and practical method of therapeutic angiogenesishas heretofore been disclosed. Thus, there is an ongoing need to developnovel therapeutic approaches for inducing angiogenesis.

SUMMARY OF THE INVENTION

The present invention provides a method for inducing angiogenesis. Themethod comprises the steps of stimulating muscles by electricalstimulation at voltages and frequencies that do not cause theircontraction. Using the method of the present invention, angiogenesis aswell as an increase in VEGF expression was observed.

In one embodiment, when cultured skeletal muscle cells were electricallystimulated at a voltage and frequency that did not cause theircontraction, vascular endothelial growth factor (VEGF) mRNA wasaugmented at an optimal-frequency stimulation. This increase of VEGFmRNA was derived primarily from transcriptional activation. Electricalstimulation increased the secretion of VEGF protein into the medium.This conditioned medium could augment the growth of endothelial cells.The effect of electrical stimulation was further confirmed in a ratmodel of hindlimb ischemia. The tibialis anterior muscle in the ischemiclimb was electrically stimulated. The frequency of stimulation was 50 Hzand strength was 0.1 V, which was below the threshold for musclecontraction. Following a 5-day stimulation, there was a significantincrease in blood flow within the muscle. Immunohistochemical analysisrevealed that VEGF protein was synthesized and capillary density wassignificantly increased in the stimulated muscle. Rats tolerated thisprocedure very well, and there was no muscle contraction, muscle injury,or restriction in movement.

Accordingly, an object of the present invention is to provide a methodfor upregulating the VEGF in muscles by electrical stimulation.

Another object of the present invention is to provide a method forinducing angiogenesis by electrical stimulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are representations of the expression of VEGF mRNA incultured smooth muscle cells, skeletal muscle cells and flk-1 mRNA inBCECs. Cells were electrically stimulated for 24 hours at indicatedfrequencies, total RNA was extracted, and Northern blotting for VEGFmRNA or flk-1 mRNA was performed for murine skeletal muscle cell line,C2C12 cells (FIG. 1A), rat aortic smooth muscle cells (FIG. 1B), andBCECs (FIG. 1C).

FIG. 2 is a representation of the analysis of stability of VEGF mRNA inC2C12 cells. Total RNA was extracted at indicated time points. Northernblotting and quantification of VEGF mRNA was performed. Percentremaining RNA was plotted as a function of time for electricalstimulation alone (∘); actinomycin D without electrical stimulation (□);and actinomycin D with electrical stimulation (□).

FIG. 3 is a representation of the time course of induction of VEGF mRNAby transient electrical stimulation. C2C12 cells were exposed to 2 hoursof electrical stimulation and total RNA was extracted at indicated timepoints after transient electrical stimulation. “Control” indicatesbefore stimulation, and “Restimulation” indicates that cells werestimulated a second time for 2 hours at 22 hours after firststimulation. Total RNA was extracted at 22 hours after secondstimulation.

FIGS. 4A-4C is a representation of synthesis, secretion and biologicalactivity of VEGF protein in conditioned medium collected duringcontinuous electrical stimulation, and secreted VEGF protein in mediumdetermined by ELISA. FIG. 4A represents rat aortic smooth muscle cells(solid columns) and C2C12 cells (open columns) exposed to indicatedfrequencies for 24 hours. In FIG. 4B, C2C12 cells were electricallystimulated and conditioned medium was collected at indicated time points(solid columns) or C2C12 cells without electrical stimulation (opencolumn). In FIG. 4C, conditioned medium of C2C12 cells with or withoutelectrical stimulation was collected and the effect on cell numberdetermined on BCECs.

FIG. 5 is a representation of the blood flow in rat tibialis anterior(TA) muscles on 7 and 14 days post operation. Electrical stimulation wascontinued from postoperative day 8 to day 12. Values are expressed as apercentage of blood flow in experimental muscles versus contralateralmuscles. At postoperative day 7, blood flow in femoral artery (FA)excision alone (□) and FA excision with electrical stimulation (∘) weresignificantly lower than in sham-operated limbs (□) (*P<0.01). Atpostoperative day 14, values in electrically stimulated in sham-operatedlimbs were significantly higher than those in FA excision alone(**P<0.01), and there was no significant difference in blood flowbetween stimulated and sham-operated limbs. All values are given as mean±SD.

FIG. 6A-C is a representation of immunohistochemical staining for VEGFprotein in rat TA muscles for contralateral TA muscle (A), TA musclesafter 5 days of electrical stimulation (B), and unstimulated TA musclewith FA excised (C).

FIG. 7 is a representation of capillary density in rat TA muscles forcontralateral, stimulated, unstimulated and sham-operated.

FIGS. 8A-8B is a representation of photomicrographs of muscles from lane2 (FIG. 8A) and lane 3 (FIG. 8B) from FIG. 7.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a simple and practical method forangiogenesis. The method of the present invention comprises the step ofelectrically stimulating the muscle at low voltage.

The term “low voltage” as used herein means, voltage that does notresult in contraction of the muscle.

Electrical stimulation of cells may be carried out by standardtechniques known in the art. For example, cells in culture may beelectrically stimulated by immersing electrodes into the culture mediaand then passage of an electrical current. The electrodes need not touchthe cells. For in vivo applications, electrodes may be implanteddirectly into the desired muscle. In the case of TA muscle, theelectrodes may in implanted into the fascia of the TA muscle, tunneledsubcutaneously and exteriorized at an appropriate location such as thescapulae and connected to a pulse generator. For application to themyocardium, electrodes may be applied via catheters or via implantedstimulators.

The electrical stimulation should be such that it does not inducecontraction of the muscle. Although the electrical frequency and voltageuseful for the method of the present invention varies with the celltype, it is preferable to use a frequency below 100 Hz. For the rat TAmuscle, it was observed that 0.1V caused angiogenesis. This is about 10%of the threshold of muscle contraction. These values can easily bemodified by those skilled in the art for therapeutic angiogenesis inindividuals in need of treatment. An example of a useful frequency andvoltage for smooth muscle is 25 Hz at 1V in vitro and for skeletalmuscle is 50 Hz at 1.0V in vitro and 50 Hz at 0.1V in vivo.

The method of the present invention can be used to promote angiogenesisin damaged or ischemic muscle tissues. The muscles wherein angiogenesiscan be stimulated include smooth, skeletal and cardiac.

The invention will be more clearly understood by the following exampleswhich are intended to be illustrative and are not to be construed asrestrictive.

EXAMPLE 1

This embodiment describes the induction of VEGF mRNA by electricalstimulation. To illustrate this embodiment, in vitro systems were usedand VEGF, Flk-1 and GAPDH mRNA were evaluated. Flk-1 is a VEGF receptorand GAPDH was used as a housekeeping gene. Murine skeletal muscle cellline C2C12 cells were obtained from Riken Cell Bank; rat aortic smoothmuscle cells from Hanno Research Center, Taiho Pharmaceutical Co Ltd;and human pulmonary artery smooth muscle cells from Kurabo. Cells wereroutinely cultured on plastic dishes in Dulbecco's Modified Eagle'sMedium (DMEM, Nissui Pharmaceutical Co, Ltd) containing 10% fetal calfserum (FCS, Summit Biotechnology). Bovine capillary endothelial cells(BCECs) (obtained from Dr. T. Tamaoki, Kyowa Hakko Kogyo Co, Ltd) weregrown in DMEM containing 10% FCS by standard methods such as describedby Sato (1991, Biochem Biophys Res Commun., 180:1098-1102). All primarycultures of passage <6 were used in following experiments.

Before conducting experiments on electrical stimulation, cells werepreincubated for 24 hours in DMEM containing 0.1% BSA. Confluentcultures were electrically stimulated according to the method describedby Brevet et al., (1976, Science, 193:1152-1154). Briefly, electrodeswere immersed in the culture medium and electric current of 1.0Vstimulus strength was passed at indicated frequencies (Hz) for theindicated time.

To analyze RNA, Northern blot analysis was carried out by standardmethods such as described by Iwasaka et al. (1996, J Cell Physiol.,169:522-531). Briefly, total RNA was extracted by the AcidGuanidium-Phenol-Chloroform method and fractionated on a 1% agarose gelcontaining 2.2 mol/L formaldehyde. The blots were then prepared bytransfer onto a nylon filter (Hybond N⁺, Amersham). The filter washybridized with a ³²P-labeled probe in hybridization solution for 24hours at 42° C. After the hybridization, the filter was washed in 2×SSCand 0.1% SDS at 60° C. and then in 0.2×SSC and 0.1% SDS at 60° C.Autoradiography was carried out on an imaging plate, and autoradiogramswere analyzed with an image analyzer (FLA 2000 Fuji). The amount of VEGFmRNA was corrected for loading differences by the amount of ribosomalRNA. Human GAPDH cDNA templates were prepared by standard methods(Iwasaka et al., 1996, J. Cell Physiol, 169:522-531). Flk-1 and VEGFcDNA templates were prepared by reverse-transcription polymearse chainreaction using the following primer pairs: Flk-1 sense (SEQ ID NO:1) andantisense (SEQ ID NO:2); VEGF sense (SEQ ID NO:3) and antisense (SEQ IDNO:4).

To determine whether electrical stimulation affects the expression ofVEGF mRNA, cultured skeletal muscle cells were exposed to electricalpulse stimulation for 24 hours at various frequencies and 1.0 Vstrength, which did not cause their contraction or interfere with theirspontaneous contraction. Northern blot analysis revealed that VEGF mRNAwas augmented at specific frequencies. In murine skeletal muscle cellline C2C12 cells, the expression of VEGF mRNA was significantlyaugmented at the narrow range of frequencies around 50 Hz. (FIG. 1A).This frequency of electrical stimulation augmented the expression ofVEGF mRNA in primary culture of rat skeletal muscle cells as well (datanot shown). Although 50-Hz electrical stimulation seemed to be optimalfor the expression of VEGF mRNA in skeletal muscle cells, it did notaugment the expression of VEGF mRNA in rat aortic smooth muscle cells(FIG. 1B) or human fibroblasts (data not shown). Instead, the expressionof VEGF mRNA was augmented at 24-Hz stimulation in rat aortic smoothmuscle cells (FIG. 1B) as well as human pulmonary artery smooth musclecells (data not shown). A time-course experiment revealed that VEGF mRNAwas increased 4.8-fold in skeletal muscle cells and 4.5-fold in smoothmuscle cells at the 8-hour time point at the proper frequency. Toexclude the possibility that substances that might be released from theelectrodes into the medium affected the expression of VEGF mRNA, themedium was electrically agitated and exposed to the cells. Noaugmentation of the expression of VEGF mRNA by this procedure wasobserved (data not shown). The expression of VEGF receptor-2 (KDR/Flk-1)mRNA in BCECs was not affected by electrical stimulation at variousfrequencies (FIG. 1C).

The level of expression of VEGF mRNA was determined by the transcriptionrate of VEGF gene and/or the stability of VEGF mRNA. An inhibitor of RNAsynthesis, antinomycin-D (Act-D) was used to evaluate the stability ofVEGF mRNA. Four μmol/L of Actinomycin D was added before electricalstimulation. Since Actinomycin D inhibits de-novo synthesis of mRNA thehalf-life of the previously synthesized mRNA could be studied. Total RNAwas extracted at indicated time points and Northern blotting wasperformed. As shown in FIG. 2, the half-lives of VEGF mRNA, which werecalculated by drawing the best-fit linear curve on a log-linear plot ofthe percentage of RNA remaining versus time in Act-D-treated cells, were0.99 hour with electrically stimulation and 1.12 hour without electricalstimulation. Thus, electrical stimulation did not affect the stabilityof VEGF mRNA. Although not intending to be bound by any particulartheory, these results suggest that the augmentation of VEGF mRNA byelectrical stimulation was at the transcriptional level.

Next, the effect of transient electrical stimulation was examined. Cellswere exposed to 2 hours of electrical stimulation, and then the totalRNA was harvested at 22 hours after the transient stimulation. Theresults showed that the induction of VEGF mRNA was observed 22 hoursafter the transient-stimulation, and the level of its expression wasalmost identical to that of the continuous electrical stimulation for 24hours (FIG. 3). Because VEGF mRNA returned to the basal level by 46hours after a 2-hour electrical stimulation, the second stimulationcould increase VEGF mRNA to a level comparable to the first stimulation.Thus, the transient electrical stimulation was equally effective and wasrepeatable.

Example 2

This embodiment demonstrates that VEGF protein is synthesized andsecreted into the medium upon electrical stimulation of the muscleaccording to the present invention. To illustrate this embodiment, C2C12cells in DMEM containing 0.1% BSA were electrically stimulated for 24hours at 1.0 V and 50 Hz, and the medium was collected. VEGF protein inthe medium was measured by Quantikine-M ELISA kit (R&D Systems)according to the manufacturer's protocol.

Conditioned medium was collected from BCECs (5×10⁴) were plated onplastic dishes in DMEM with 10% FCS and incubated for 6 hours to allowattachment to the dish. The culture medium was replaced with either 100%conditioned medium, fresh medium (DMEM containing 0.1% BSA), or freshmedium supplemented with 10 ng/mL of recombinant human VEGF. Cellnumbers were counted after 48 hours of incubation.

The synthesis and secretion of VEGF protein into the medium was analyzedby ELISA. VEGF in the medium of both C2C12 cells and rat aortic smoothmuscle cells was increased by electrical stimulation (FIG. 4A). Thesestimulatory effects were observed to increase VEGF mRNA at the samespecific frequencies in these cells. The elevation of VEGF protein inthe medium was observed as early as 12 hours and reached its peak at 48hours (FIG. 4B). The conditioned medium of electrical stimulationaugmented the growth of BCECs as much as the medium supplemented with 10ng/mL of human recombinant VEGF (FIG. 4C). Although proteins other thanVEGF might also be responsible for the growth of BCECs, theconcentration of VEGF in conditioned medium determined by ELISAsuggested that most of the effects on the growth of BCECs were derivedfrom VEGF and that electrical stimulation stimulated not only geneexpression but also post transcriptional events in the synthesis ofVEGF.

EXAMPLE 3

This embodiment demonstrates that electrical stimulation inducesangiogenesis. This embodiment is illustrated by using a rat model ofischemic hindlimb. The parameters used to assess angiogenesis wereimmunohistochemical localization of VEGF, capillary density and bloodflow. The statistical significance of differences in the results wasevaluated by use of unpaired ANOVA, and a value of P<0.05 was acceptedas statistically significant.

Male Sprague-Dawley rats (300 to 350 g body weight) were anesthetizedwith light ether sedation and subcutaneous injection of pentobarbitalsodium (50 mg/kg) (Nembutal, Abbott Laboratories). The operation forhindlimb ischemia was performed according to the method described byTakeshita et al. (1996, Lab Invest., 74:1061-1065). Briefly, the leftfemoral artery (FA) was completely excised from its proximal origin tothe point distally at which bifurcates into the saphenous and poplitealarteries. After 1 week was allowed for recovery from the operation,blood flows of the bilateral tibialis anterior (TA) muscles weremeasured by the hydrogen gas clearance technique originally described byAukland et al. (1964, Circ Res., 14:164-187) and modified by Hori et al.(1996, Int J Cancer., 65:360-364). Thereafter, the electrodes (Electrodefor Functional Electrical Stimulation (FES), Nihonseisen) were implantedonto the facia of the left TA muscle, tunneled subcutaneously andexteriorized at the lever of the scapulae (Hang et al., 1995, Am. J.Physiol., 269:H1827-H1831) and connected to a pulse generator(PulseCure, OG Giken). Electrical stimulation was started on the dayafter electrode implantation and continued for 5 days with a 0.3-msstimulus width, 50-Hz stimulus frequency, and 0.1-V stimulus strength,which was far below the threshold of TA muscle contraction. Thethreshold of TA muscle contraction in this experimental conditiondetected by electromyogram was 1.1±0.2 V (data now shown). One day afterthe period of electrical stimulation, animals were anesthetized, bloodflow was measured, and then the animals were killed for the procurementof bilateral TA muscles for further evaluations.

Animals were divided into 3 groups. In the first group (5 animals), TAmuscles were continuously stimulated for 5 days after the dissection andexcision of the left FA. In the second group (4 animals), electrodeswere implanted after the dissection and excision of the left FA, but TAmuscles were not electrically stimulated. The third group (6 animals)received a sham operation of the left FA and no electrical stimulationdespite the implantation of electrodes. The contralateral hindlimb ofeach animal served as the control.

Blood flow was measured in TA muscles of ischemic and contralaterallimbs. The percentage of blood flow in experimental muscles versuscontralateral muscles is shown in FIG. 5. Blood flow in the ischemiclimb (□) on day 7 after the left FA excision was significantly lowerthan that in the contralateral limb as well as in the sham-operatedanimals (□). Whereas blood flow in the ischemic limb on day 14 after theleft FA excision did not increase without any stimulation (□),continuous electrical stimulation significantly increased blood flow inthe TA muscle of the ischemic limb (∘). In addition, rats tolerated thisprocedure very well, and there was no muscle contraction, muscle injury,or restriction in movement.

For detection of VEGF protein, muscle specimens were fixed with 10%formaldehyde, preincubated with 1% bovine serum albumin (BSA) for 30minutes, and then incubated with rabbit polyclonal anti-VEGF antibody (1μ/mL) (Santa Cruz Biotechnology) for 30 minutes at room temperature.Thereafter, the specimens were stained by the avidin-biotin complexmethod (Elite, Vector Laboratories, Inc.).

As shown in FIGS. 6A-6C, immunostaining of TA muscles with polyclonalanti-VEGF antibody revealed a significant increase of VEGF protein inthe muscle fibers of electrically stimulated TA muscles (6B) comparedwith unstimulated muscles (6C) or contralateral TA muscles (6C). Thisincrease of VEGF protein was found only in the area between theelectrodes on TA muscle.

Another parameter used to assess angiogenesis was capillary density. Thenumber of capillaries and muscle fibers were counted in fixed sectionsof at least 8 different fields, and capillary density was obtained bythe calculation of capillary number/fiber area.

As shown in FIG. 7, capillary density of the stimulated muscles (lane 2)was increased ˜2.5-fold compared with the contralateral muscles (lane 1)(P<0.01), whereas unstimulated animals (lane 3) as well as sham-operatedanimals (lane 4) had no significant difference between ischemic andcontralateral muscles (FIG. 7). Representative images of the stimulated(A) and unstimulated (B) muscle from FIG. 7 are shown in FIG. 8, againdemonstrating an increase in vasculature following electricalstimulation.

The data presented in this application demonstrates that electricalstimulation at low voltages that does not cause contraction, inducesangiogenesis. While the invention has been described in detail, theabove examples are illustrative and are not to be construed asrestrictive. Various modifications of the present embodiments that areobvious to those skilled in the art are intended to be within the scopeof the invention described herein.

1. A method for inducing angiogenesis in a muscle tissue comprising thesteps of applying electrical voltage to one or more areas of the tissue,wherein the electrical voltage does not induce contraction of musclecells within the muscle tissue, and wherein angiogenesis is inducedafter application of the electric voltage.
 2. The method of claim 1,wherein the voltage is 0.1V at a frequency of 50 Hz.
 3. The method ofclaim 1, wherein the muscle cells are smooth muscle cells.
 4. The methodof claim 1, wherein the muscle cells are skeletal muscle cells.
 5. Themethod of claim 1, wherein the muscle cells are cardiac muscle cells. 6.A method for increasing VEGF in a muscle cell comprising the step ofapplying electrical voltage to the muscle cell, wherein the electricalvoltage does not induce contraction of the muscle cell and wherein VEGFis increased after application of the electrical voltage.
 7. The methodof claim 6, wherein the voltage is 0.1V applied at a frequency of 50 Hz.8. The method of claim 6, wherein the muscle is a smooth muscle.
 9. Themethod of claim 6, wherein the muscle is a skeletal muscle.
 10. Themethod of claim 6, wherein the muscle is a cardiac muscle.